The present invention relates to the recovery and purification of enzymes from fermentation preparations, and the purified enzyme product that results therefrom.
The use of enzymes in detergents is well known. Generally, enzymes used in detergents have primarily been the alkaline stable proteases, lipases and alpha-amylases. Of the alkaline proteases, serine proteases derived from Bacillus species, namely Bacillus subtilis, Bacillus licheniformis, and alkalophilic Bacillus bacteria have been widely used in detergent formulations. (Starace, C. and Barford, H. C., Encyclopedia Chem. Technol. 9, pp. 138-148 (1980); Koki Horikoshi and Terahiko Akika, A New Microbial World, Springer-Verlag, New York, p. 93 (1982)).
Enzymes constitute only a small portion of most liquid detergent formulations. Thus, it is necessary to produce fairly concentrated enzyme preparations. Enzyme concentrates are traditionally prepared by removing the water from aqueous solutions of the enzymes using conventional methods such as ultrafiltration and evaporation.
Inorganic salts such as ammonium sulphate and sodium sulphate have been used extensively to precipitate enzymes from aqueous solution at both the laboratory and commercial levels. (Dixon, M. and Webb, E. D., Enzymes, Academic Press, N.Y., pp. 39-41 (1964), Curline, Methods of Plasma Protein Fractionation, Academic Press, New York (1980)). The widespread use of these salts on a large scale, however, can pose environmental problems and complicate waste water treatment. In fact, many countries in Europe have already restricted large scale industrial use of these salts. Organic solvents such as ethanol and acetone are also used as precipitants (Dixon and Webb, Enzymes, supra, pp. 37-39; Bauer et al,. J. Chem., 5(3), pp. 117-20 (1967)), however, their use has been limited because of cost and concern for safety.
The color and odor of enzymes can adversely effect the quality of the detergent formulations in which they are incorporated. This necessitates the removal of pigments from the enzyme concentrate, which pigments are believed to be a part of an enzyme-pigment complex. Dixon, M. and Webb, E. C., Enzymes, supra, reported solvent precipitation methods to remove pigment from protease solution. This method, however, resulted in poor product yield. Absorption of pigments with activated carbon from aqueous enzyme concentrate is generally practiced in industrial applications, however, material loss, high cost and waste disposal present major drawbacks.
It is desirable that enzyme preparations for detergent applications be free from components which can cause undesirable color, haze, instability and allergic activity in the final product. These components may be derived from the microorganism themselves or from residual fermentation raw materials. In preparations of gram positive Bacilli, cell wall anionic polymers, peptidoglycans, galactosyl polymers, and other polysaccharide contaminants become solubilized during cell growth due to cell wall turnover. The presence of these bacterial cell wall polymers in enzyme preparations can cause several undesirable effects including an increase in the allergenicity, a decrease in enzyme stability by binding cations, e g., Ca.sup.++, and may cause haze formation in detergent formulations.
In U.S. Pat. No. 4,659,667 a method for crystallization of an enzyme is disclosed wherein the pH of the super saturated enzyme solution is adjusted to its isoelectric pH.
In U.S. Pat. No. 4,699,882 a method for crystallization of glucose isomerase using ammonium and magnesium sulphate is disclosed.
In Patent Cooperation Treaty ("PCT") Application No. W089/05863 published Jun. 29, 1986, a method for separation of the galactosyl polymer associated with allergenic activity from protease preparations is described using ion-exchange chromatography.
In U.S. Pat. No. 5,041,377, a method for obtaining crystalline subtilisin is described where in subtilisin derived from Bacillus subtilis and Bacillus amyloliquifaciens is crystallized by the addition of a halide salt (sodium chloride and potassium chloride) to an alkaline protease solution at low temperature.
In PCT Application WO 91/09943 a method for the crystallization of enzymes is described where an aqueous enzyme containing liquid with a relatively high enzyme purity and with a concentration of pure enzyme protein of at least 5 g/l is used as a starting material, and a crystallization agent which is an easily soluble salt of the non-halide type, such as Na, K, Ca, or Mg formate, acetate or nitrate, is added to the starting material. In the examples using proteases, the starting material is prepared by sodium sulphate precipitation.
None of the patents, patent applications or publications described above provide the important advantages of a simple and efficient method of preparing a purified enzyme preparation free from contaminants wherein a high yield of purified enzyme is obtained, and the use of inorganic salts or other nonbiodegradable compounds is avoided.